KR20220046735A - An inhalation-type therapeutic agent using pulmonary surfactant for coronavirus treatment - Google Patents

Abstract

The present invention provides a method for preparing an inhaled coronavirus therapeutic agent so that the hydrophobic drug Zafirlukast, which is prescribed as an oral asthma treatment, is loaded into a biocompatible pulmonary surfactant containing hydrophobic lipids and can be effectively delivered to the respiratory tract.
In addition, the present invention provides a method for loading zafirlukast drug in a surfactant using a relatively inexpensive pharmaceutical surfactant that can replace expensive waste surfactant.
As described above, according to the method for manufacturing an inhaled coronavirus therapeutic agent using the pulmonary surfactant of the present invention, the side effects and toxicity of the therapeutic drug spread to other parts of the body are minimized, and the dosage of the therapeutic drug delivered to the treatment site is optimized. , while reducing the toxicity and side effects of the drug, it can maximize the efficacy of the treatment of coronavirus, and it has the advantage of reducing the production cost of the therapeutic agent by reducing the manufacturing process cost.

Description

Inhaled coronavirus treatment using pulmonary surfactant {AN INHALATION-TYPE THERAPEUTIC AGENT USING PULMONARY SURFACTANT FOR CORONAVIRUS TREATMENT}

The present invention relates to an inhaled coronavirus therapeutic agent and a method for manufacturing the same.

Remdesivir is a drug developed by multinational pharmaceutical company Gilead as a treatment for Ebola. Remdesivir, originally developed as a treatment for Ebola, is effective in patients with severe COVID-19 through the drug re-creation process, and is being administered as a treatment in each country. Since remdesivir has a similar structure to Corona 19 and nucleic acid (adenine), it uses a mechanism to treat the virus by deceiving the virus that has invaded the human body. In other words, remdesivir has a virus-like structure by modifying some of the molecular structure of the virus, but is transformed as soon as it enters the virus cell and is involved in the synthesis of viral RNA by polymerase. As a result, remdesivir has a therapeutic effect through a mechanism that prevents the virus from further multiplying. In this way, research on drug re-creation is being conducted focusing on drugs that have been proven effective in treating other viruses, such as using existing RNA virus therapeutics to treat COVID-19.

On the other hand, when Zafirlukast, which is prescribed as an oral asthma treatment, is administered to the respiratory tract, an appropriate amount of the drug can be effectively delivered to the lungs, and at the same time, toxicity and side effects of the drug can be minimized, thereby maximizing the therapeutic efficacy of the Corona 19 virus. can do it

In this way, it is possible to develop a technology for manufacturing an inhalation formulation that can be administered through the respiratory tract by loading the COVID-19 treatment, which is being developed for oral or intravenous use, such as remdesivir, in a pulmonary surfactant that has a drug delivery function.

Meanwhile, among lung cancers, adenocarcinoma accounts for about 40% of all lung cancers, mainly occurs in the alveolar region, and often occurs in type II alveolar cells. These type II alveolar cells (alveolar type II cells) function to secrete and store a bio-derived pulmonary surfactant, and the pulmonary surfactant plays a role in regulating the tension of the lungs during respiration. .

These bio-derived lung surfactants are composed of lipids and membrane proteins, and when the therapeutic agent is delivered to target cells using the lung surfactant, it secretes and stores the lung surfactant. It can effectively target an anticancer agent to II alveolar type II cells. On the other hand, a technology for encapsulating an anticancer agent in a liposome prepared with such a lung surfactant has also been developed.

The development of a technology to effectively deliver the lung surfactant to the lungs by combining it with a therapeutic agent for the treatment of the Corona 19 virus is a very urgent technical task at present.

Such a pulmonary surfactant may form a complex with a therapeutic drug, and may be in a form in which the pulmonary surfactant and the therapeutic drug are combined through a covalent bond.

On the other hand, the pulmonary surfactant may be a lipoprotein complex produced in type II alveolar cells, and such pulmonary surfactant may be collected and purified from mammalian lungs.

Since this natural pulmonary surfactant is secreted and stored in the type II alveolar cells, the therapeutic drug combined with the pulmonary surfactant can be effectively delivered to a target treatment site.

The term lipid in the lipoprotein complex refers to a naturally occurring, synthetic or semi-synthetic (ie, modified natural) compound that is generally amphiphilic. Lipids typically include a hydrophilic component and a hydrophobic component. Exemplary lipids may include phospholipids, fatty acids, fatty alcohols, triglycerides, phosphadides, oils, glycolipids, fatty alcohols, waxes, terpenes, and steroids. The term "semi-synthetic (or modified natural)" refers to a natural compound that has been chemically modified in some way.

On the other hand, phospholipids include natural and / or synthetic phospholipids, phospholipids that can be used include phosphatidylcholine (saturated and unsaturated), phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, sphingolipids, diacylglycerol Ride, cardiolipin, ceramide and cerebroside and the like may be included.

Further, examples of fatty acids and fatty alcohols may include sterol, palmitic acid, cetyl alcohol, lauric acid, myristic acid, stearic acid, phytanic acid, dipalmitic acid, and the like. Specific examples of fatty acids include palmitic acid, and specific examples of fatty acid esters include methyl palmitate, ethyl palmitate, isopropyl palmitate, cholesteryl palmitate, palmityl palmitate, sodium palmitate, potassium palmitate and Tripalmitin, etc.

On the other hand, the lung surfactant includes a membrane protein, the membrane protein being at least one natural surfactant polypeptide selected from the group consisting of SP-A, SP-B, SP-C and SP-D; a portion thereof or a mixture thereof. Exemplary peptides may comprise at least about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acid segments of a native surfactant polypeptide.

In addition, the polypeptide can remain cytoplasmically intact, the cells are harvested, lysed, and the protein isolated from the cell lysate. The surfactant polypeptide and surfactant lipid interact by hydrostatic interaction. Charged amino acids interact with the polar head groups of lipids, and hydrophobic amino acids interact with phospholipid acyl side chains.

For example, SP-B and SP-C are hydrophobic proteins. Both SP-B and SP-C bind preferentially to anionic lipids (eg, phosphatidylglycerol (PG), but not DPPC). SP-A and SP-D are hydrophilic proteins and interact with a wide range of amphiphilic lipids, including glycerophospholipids, sphingophospholipids, sphingoglycolipids, lipid A and lipoglycans. SP-A binds to DPPC. As an example, hydrostatic interaction of KL4, an SP-B mimetic, with lipids in natural surfactants or lipids contained in surface active agents is observed. For example, a lysine residue in the KL4 peptide interacts with the charged head group of DPPC, and a hydrophobic leucine residue interacts with the phospholipid acyl side chain of phosphatidylglycerol.

The drug currently being used as a treatment for COVID-19 is Remdesivir. There are pharmacokinetic studies showing that remdesivir is not suitable for oral administration due to its first-pass effect, and slow and volatile release from the muscle is also not suitable for intramuscular injection. Therefore, it is currently known that only intravenous injection is possible, and when administered intravenously, it is distributed to various organs such as the kidneys, liver, and aorta and reaches the lungs. As such, when administered intravenously, the amount of the drug that is lost is greater than the amount administered, so that the amount of the drug that reaches the treatment target site is substantially reduced. In addition, since the distribution is made systemically, unnecessary side effects also occur, which causes a problem that requires additional treatment for side effects in the future.

There is a need to develop a manufacturing method that can mount Zafirlukast, a candidate material for treatment of COVID-19, in such an appropriate lung surfactant as efficiently as possible. In addition, technology development is required for a technology capable of manufacturing Zafirlukast into an inhalation formulation.

The problem to be solved by the present invention is to mount the hydrophobic drug Zafirlukast, which is prescribed as an oral asthma treatment, in a biocompatible pulmonary surfactant containing hydrophobic lipids to effectively deliver it to the respiratory tract. To provide a manufacturing method.

In addition, another problem to be solved by the present invention is to provide a method for loading the drug zafirlukast in a surfactant using a relatively inexpensive pharmaceutical surfactant that can replace expensive waste surfactant.

It is intended to determine the efficacy of the inhaled coronavirus treatment provided by the present invention by conducting animal experiments.

Preparing a first solution in which the present invention coronavirus therapeutic agent (zafirlukast) is dissolved;

Preparing a second solution in which the lung surfactant is dissolved;

mixing the first solution and the second solution, and drying the mixed solution to form a film; and

It provides a method for manufacturing an inhaled coronavirus treatment using a lung surfactant, comprising the step of treating the film with distilled water for hydration to prepare a complex in which the corona treatment agent is encapsulated.

At this time, the hydration (hydration) may be performed at a temperature of 40 to 90 ℃, more preferably performed at a temperature of 60 to 70 ℃.

In addition, after performing all of the above steps, the step of adjusting the diameter of the coronavirus treatment may be further performed, and an extrusion kit may be used for the purpose of adjusting the diameter.

And, the coronavirus therapeutic agent prepared by encapsulating zafirlukast in liposomes made of pulmonary surfactant can be effectively delivered to alveolar cells, and has low toxicity and excellent structural stability.

On the other hand, the present invention provides a method for preparing a sterile composition for administering a therapeutic agent for coronavirus as an aerosol, and the sterile composition may include an active agent having low water solubility, a nonionic surfactant component and a phospholipid component.

The sterile composition by aerosol administration according to the present invention is suitable for topical or oral mucosal administration as well as oral or nasal inhalation.

The sterilization composition of the present invention comprises a poorly water-soluble active agent and a nonionic surfactant component, wherein the nonionic surfactant component is tyloxapol, polysorbate, vitamin E TPGS, macrogol-hydroxy stearate, and a phospholipid component. It may include one or more components selected from, and the active agent is not a surfactant and can be dissolved in unilamellar liposomes.

In addition, the phospholipid component may include one or more components selected from the group consisting of zwitterionic phospholipids, saturated phospholipids, hydrogenated phospholipids, and pure phospholipids.

And, the weight ratio of the nonionic surfactant component and the phospholipid component may be in the range of about 5:1 to about 1:20.

On the other hand, the sterilizing composition of the present invention further comprises one or more excipients selected from the group consisting of acids, bases, buffers, osmotic agents, stabilizers, antioxidants, taste masking agents, flavoring agents, sweeteners, ionic surfactants, thickeners, and coloring agents can do.

The antioxidant may be one or more components selected from the group consisting of vitamin E acetate, EDTA (di) sodium salt, and mixtures thereof.

In addition, the sterilization composition according to the present invention may be in the form of an aqueous liquid.

The sterile composition of the present invention may have an average particle size of the liposomes and a polydispersity index of about 0.3 or less, and the particle size is in the range of about 10 to 100 nm, and does not include solid particles that are precipitated.

The sterile composition of the present invention may comprise from about 0.01 to about 5.0 weight percent of a non-ionic surfactant component and from 0.5 to about 5 weight percent of a phospholipid component.

On the other hand, when the sterilization composition of the present invention is in a crystal liquid state, a surface tension of about 30 to about 75 mN/m and a kinematic viscosity of 0.8 to about 3.0 mPas, a pH in the range of about 4 to 8, about 200 to 500 mOsmol / kg It may exhibit osmotic pressure characteristics.

In addition, the sterile composition of the present invention may include (a) a cosolvent 　 and/or (b) a preservative, and may contain from about 0.001 weight % to about 1 weight % of a sparingly water-soluble active agent.

On the other hand, the sterile composition of the present invention may include one or more components selected from the group consisting of cell proliferation inhibitors such as antibacterial and antifungal.

The sterile composition of the present invention may be a dry solid composition obtained by freeze-drying an aqueous liquid composition.

On the other hand, the sterile composition of the present invention can be prepared through the following steps.

(a) providing ingredients;

(b) preparing an aqueous liquid composition from the components provided in step (a);

(c) sterile filtration of the composition obtained in step (b); and

(d) filling the sterile filtered composition of step (c) into a sterile container under aseptic conditions.

In the above process, at least one of the components provided in step (a) may be sterilized, and step (b) may include a sub-step of homogenization under increased pressure, sonication, and/or heating to 45° C. or higher. can

In addition, the sterile container of step (d) may be a plastic vial prepared by an aseptic blow-peel-seal process.

According to the method for manufacturing an inhaled coronavirus therapeutic agent using a pulmonary surfactant according to the present invention, the side effects and toxicity of the therapeutic drug spread to other parts of the body are minimized, and the dosage of the therapeutic drug delivered to the treatment site is optimized, While reducing the toxicity and side effects of the drug, it is possible to maximize the efficacy of the coronavirus treatment, and it has the advantage of reducing the production cost of the treatment by lowering the manufacturing process cost.

1 is a flowchart illustrating the manufacturing process of the drug-loaded lung surfactant-based particles according to the present invention.
2 is a graph of a calibration curve measuring the correlation between loading of pulmonary surfactant while changing the concentration of zapirukast according to the present invention.
3 is a graph showing how much the concentration of zapyrukast is doubled and loaded in the pulmonary surfactant is measured.

<Preparation Example 1> Preparation of drug-encapsulated lung surfactant-based particles

Zapirukast is a drug used for the prevention and continuous treatment of asthma by inhibiting CYP2C9. A brief overview of the mechanism of action of zapirukast is as follows. Arachidonic acid is metabolized to prostaglandins and thromboxanes by cyclooxygenase and to leukotrienes by 5-lipoxygenase.

In addition, Leukotrienes is sequentially metabolized to LTC, LTD ₄ , and LTE, which is called Cysteinyl leukotrienes (CysLT). Cysteinyl leukotrienes is a slow-acting substance in the anaphylaxis reaction and causes inflammatory responses in the airways, bronchial smooth muscle contraction, and edema. Zapirukast (and montelukast) blocks these receptors and suppresses the inflammatory response.

[Formula 1]

However, according to the results of a randomized screening trial reported in the medical journal 'Journal of Family Practice 2001:50:595-602', fluticasone, an inhaled corticosteroid, was not well regulated by short-acting beta-2 coordination. It has also been reported that it is more effective than zapirukast, a leukotriene modulator, for asthma patients.

Therefore, although zapirukast is used as an asthma treatment in the present invention, it is not necessarily limited to only zapirukast, and other asthma treatment agents such as fluticasone can also be used as drugs.

In the present invention, using zafirlukast, which is effective for symptom relief and treatment of asthma, as a coronavirus treatment, drug-encapsulated lung surfactant-based particles were prepared through the following process.

Zapyrukast powder was dissolved in methanol at a concentration of 10 mg/ml (first solution).

In a solution in which chloroform and methanol were mixed in a volume ratio of 2:1 (v:v), waste surfactant powder (manufacturer: Mitsubishi / product name: Surfacten) was dissolved at a concentration of 10 mg/ml (second solution) ).

The first solution and the second solution were mixed so that the waste surfactant and zapyrukast could be mixed in a ratio of 20:1 (w:w) by mass, and the mixed solution was put into a glass bottle and dried. At this time, if necessary, the mass ratio of mixing the lung surfactant and zapirukast can be adjusted in an appropriate range for optimal drug loading.

Then, the dried waste surfactant was hydrated with distilled water to a concentration of 3 mg/ml. However, during hydration, the experiment was carried out on a hot plate, and the temperature was maintained at 60-70 °C. The particles produced during the hydration process were made to have an average diameter of 400 nm using an extruder kit.

Then, the drug that was not encapsulated in the particles was separated for 12 hours through a dialysis technique using a 100 kDa membrane. Thus, a drug (zapirukast) was prepared in the encapsulated pulmonary surfactant particles (see [Fig. 1])

When the drug-encapsulated lung surfactant-based particles are prepared through the process of mixing the lung surfactant and the drug in a 20:1 ratio by mass as described above, the drug in the final manufactured particle is about 1 compared to the mass of the particle itself. As much as % by weight of drug can be encapsulated. That is, in Preparation Example 1, 1 part by weight of the drug was encapsulated with respect to 100 parts by weight of the particle itself.

On the other hand, using DiI, which is a very hydrophobic, poorly water-soluble dye, in order to observe intracellular uptake, which will be described later, the dye-encapsulated lung surfactant-based particles were prepared through the following process.

The red dye DiI was dissolved in methanol at a concentration of 1 mg/ml (first solution). In a solution of chloroform (chloroform) and methanol in a volume ratio of 2:1, a waste surfactant powder was dissolved at a concentration of 10 mg/ml (second solution). The first solution and the second solution were mixed so that the spent surfactant and the dye could be mixed in a ratio of 1000:1 by mass, and the mixed solution was put in a glass bottle and dried. The dried waste surfactant was hydrated with distilled water to a concentration of 3 mg/ml. However, during hydration, the experiment was carried out on a hot plate, and the temperature was maintained at 60-70°C. The particles produced during the hydration process were made to have a diameter of 400 nm using an extruder kit.

<Preparation Example 2> Confirmation of possibility of loading Zafirlukast drug in pulmonary surfactant

- How to measure a drug calibration curve

To measure the content of Zafirlukast loaded in the waste surfactant, the following experiment was conducted to measure the Zafirlukast calibration curve. First, 1 mg of Zafirlukast and 20 ml of methanol were put together in a glass vial and dissolved. Sonication was performed for 20 minutes so that the particles were sufficiently dissolved in the solvent. Finally, a sample of 0 to 0.05 mg/mL was prepared through a continuous dilution process using methanol as a solvent to prepare a total of 7 samples including the sample (Blank) not containing Zafirlukast.

The Zafirlukast content of the sample prepared as described above was measured through IR analysis at 220 nm, and the calibration curve at this time was shown as [Fig. 2].

As a result of checking the values of the calibration curve, the slope was found to be 0.0245 and the y-intercept was found to be 0.0263. The correlation coefficient (R2) was 0.9981. As the Zafirlukast content increased, the Zafirlukast content loaded in the waste surfactant also tended to increase linearly, showing a very high correlation.

Experiments with loading various concentrations of Zafirlukast drug in pulmonary surfactant

<Example 1>

The mixture prepared in the ratio of mass ratio Zafirlukast / waste surfactant [ratio = 0.05, w / w] in Example 1 was dried. In the case of Zafirlukast, methanol was used as a solvent, and in the case of a waste surfactant, methanol/chloroform [ratio=0.5, v/v] was used as a solvent. After adding distilled water to the dried mixture to hydrate the waste surfactant to 1 mg/ml, the particle size was adjusted on a hot plate using an extruder kit. The set temperature of the hot plate was 65°C and the actual temperature was maintained to be 50°C or higher. Thereafter, the drug not loaded on the pulmonary surfactant was separated by dialysis using a 100 kD membrane for 16 hours.

<Example 2>

The drug loading method of Example 1 was carried out in the same manner, and the mass ratio of Zafirlukast/pulmonary surfactant was mixed at a ratio of 0.1 in order to load the amount of Zafirlukast twice, and then dried and the results were confirmed. [Table 1] and [Fig. 3] below are the results for the analysis of the amount of the loaded drug in Examples 1 and 2.

[Table 1]

<Example 3>

A drug loading experiment was conducted using a surfactant component different from Examples 1 and 2, and MCT oil and Lecithin were used instead of the waste surfactant. Zafirlukast : MCT oil : Lecithin 2 : 1 : The mixture prepared in 5 ratio was dissolved in 1 mL of solvent and dried. At this time, the ratio of the drug (Zafirlukast) to the surfactant (MCT oil, Lecithin) may be changed.

Methanol/dichloromethane was used as the solvent, and a volume ratio (v/v) of 3:7 was used. The dried mixture was hydrated by adding 1 mL of distilled water, and then the drug not loaded on the surfactant was separated using a 0.22 μm filter.

As described above, the inhaled therapeutic agent manufactured through the method for manufacturing an inhaled coronavirus treatment agent using the pulmonary surfactant of the present invention can deliver the drug to the lungs more effectively than the conventional oral dosing method as a drug delivery method through inhalation. In addition, since the therapeutic agent can be directly delivered through the alveolar cells using a bio-derived or physiologically active surfactant, the amount of drug lost before reaching the desired treatment site during oral administration can be reduced, compared to oral administration. Even a small amount can increase the therapeutic effect.

On the other hand, the surfactant used in the method for manufacturing an inhaled coronavirus therapeutic agent using the lung surfactant of the present invention can be used in various ways as a carrier for delivering a specific substance (therapeutic drug), so it can be applied to various indications.

<Example 3>

Animal experiments (in vivo) were conducted with the drugs prepared in Examples 1 and 2 to confirm the efficacy of the drug. A Syrian Hamster was used, and the body weight and dynamic adverse reactions of the hamster were observed through drug administration for 14 days. The drug loaded with Zafirlukast was administered to hamsters infected with SARS-CoV-2 twice a day. Body weight and dynamic adverse reactions were observed during the survival period, and then the efficacy of the drug was confirmed by examining lung tissue, virus titer, and biomarkers.

In the present invention, it will be possible to be protected by patent rights by reinforcing the contents of the following key contents.

Claims (7)

A pharmaceutical composition comprising both a bovine and pig-derived pulmonary surfactant and a lipoprotein complex to prepare an inhalation formulation that delivers a drug directly to the lungs by breathing through the bronchi One or more drugs selected from the group consisting of Zafirlukast, Remdesivir, Dexamethasone, Nafamostat mesylate, Sulfinpyrazone, Clevudine, Hydroxychloroquine, Interferon, Atazanvir, and Cardiogreen that have been reinvented to treat coronavirus or are used as a treatment for coronavirus Pharmaceutical composition mounted on surfactant A pharmaceutical composition that can be used as an inhalation antiviral agent, characterized in that it is encapsulated in an amount of 0.1 to 10 parts by weight based on 100 parts by weight of a liposome made of a lung surfactant and a surfactant as a therapeutic agent for coronavirus As the loading of the drug in a pulmonary surfactant or surfactant, the drug: [pulmonary surfactant and activator] mixing ratio of 20: 1 to 5: 1 , or drug: [vegetable oil (ex; MCT oil) and emulsifier ( ex: lecithin)] in a ratio of 20:1 to 5:1 , characterized in that it is prepared, a method for producing a therapeutic agent for coronavirus Pharmaceuticals that enhance emulsion stability by preparing the waste surfactant, activator, and drugs such as Zafirlukast in the form of a water-in-oil (W ₁ /O/W ₂ ) nanoemulsion in which a trapping material is added to the internal aqueous phase (W ₁ ) composition. 6. The method of claim 5,
Pharmaceutical composition comprising functional oligosaccharides such as cyclodextrin and maltodextrin in addition to the nanoemulsion formulation 7. The method according to claim 5 or 6,
A pharmaceutical composition prepared in a spray dry type by processing the nanoemulsion.

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